The DNA-dependent protein kinase (DNA-PK) is a key component of the non-homologous end-joining pathway of DNA double-strand break (DSB) repair in mammalian cells. DNA-PK activity is essential for DSB repair and VDJ recombination. DNA-PK phosphorylates a variety of substrates including its self, in vitro; however its in vivo targets remain unclear. DNA-PK is phosphorylated in vitro in response to free DNA ends. Mass spectrometry has revealed several potential phosphorylation sites present in DNA-PKcs. Previous studies in our laboratory have demonstrated that DNA-PKcs is phosphorylated at T2609 in response to DSBs and mutation of that site results in radiation sensitivity and an end-joining defect. We propose to study T2638 and T2647 phosphorylation in DNA-Pkcs and determine the role of these specific post-translational events in DNA repair and cell viability. The hypotheses to be tested are (1) DNA-PKcs will be phosphorylated at sites T2638 and T2647 in response to DSBs. (2) These sites will only be phosphorylated in G1 and early S phase and (3) the mutation of these sites will result in radiation sensitivity, an end-joining defect and a longer half-life of phospho-specific (ala) mutants at these sites. [unreadable] [unreadable]